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Manufacture of five-carbon sugars and sugar alcohols

a technology of five-carbon sugars and sugar alcohols, which is applied in the preparation of sugar derivatives, monosaccharides, enzymes, etc., can solve the problems of little effort in the opposite direction of modifying microorganisms and no method of further improving natives, so as to improve the spectrum of five-carbon carbohydrates

Inactive Publication Date: 2007-06-05
DANISCO SWEETENERS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0021]In addition to the genetic modifications, the inventors have discovered fermentation conditions that may be used to further enhance and adjust the spectrum of the five-carbon carbohydrates produced by specific hosts according to the methods of the present invention.

Problems solved by technology

However, little has been done with regard to modifying microorganisms in the opposite direction, to redirect carbon flow away from glycolysis or away from ethanol production and into the PPP, with accumulation of PPP intermediates and sugars or sugar alcohols derived therefrom.
Most teach no methods of further improving the native abilities the of microorganisms for the production of such sugars or sugar alcohols, or for broadening the spectrum of useful products produced by the fermentation.

Method used

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  • Manufacture of five-carbon sugars and sugar alcohols
  • Manufacture of five-carbon sugars and sugar alcohols
  • Manufacture of five-carbon sugars and sugar alcohols

Examples

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example 1

Cloning of the B. subtilis rpi Gene Coding for D-Ribose-Phosphate Isomerase

[0225]At the time when the work was initiated, the complete genomic sequence of B. subtilis was not yet available. Also, it was not known whether B. subtilis contained one or more D-ribose-phosphate isomerase genes (E. coli was known to contain two). Therefore, the strategy for cloning the rpi gene(s) was based on functional complementation of D-ribose-auxotrophic mutation in E. coli rather than on PCR. Presently, the preferred mode for cloning the rpi gene(s) would be to use PCR based techniques rather than the method as exemplified below. However, the method described in this example is fully adequate for practicing the present invention.

[0226]A gene library was constructed from the DNA of B. subtilis (ATCC 6051). The DNA of this strain was partially cut with the restriction endonuclease Sau3A and fragments exceeding 3 kb in size were isolated by preparative agarose gel electrophoresis. Unless indicated oth...

example 2

Construction of B. subtilis Strains Containing rpi− and tkt− Mutation

[0227]The chloramphenicol resistance gene was isolated from the plasmid pMK4 (obtained from the Bacillus Genetic Stock Center (BGSC, Ohio, USA). pMK4 was digested with DraI and EcoRI, a 1.9 kb fragment was isolated from the digest by preparative agarose gel electrophoresis and further digested with Sau3A. A purified 0.83 kb fragment from this digest was purified and treated with Klenow fragment of the DNA-polymerase I in the presence of all four deoxynucleotide triphosphates. This fragment was than ligated with the plasmid p131 (Example 1) digested with SfuI and similarly treated with the Klenow fragment. Plasmid p131-Cm2 containing the B. subtilis rpi gene disrupted by the chloramphenicol resistance gene was isolated after transformation of E. coli with this ligation mixture (FIG. 2).

[0228]p131-Cm2 was digested with EcoRI and PstI and the digest was used to transform the B. subtilis strain BD170 (trpC2, thr−5, obt...

example 3

Construction of D-ribulose-producing B. subtilis Strains

[0231]Chromosomal DNA was isolated from the strain GX1 by the method referred to in Example 2 (except that Na-sarcosyl used in the original protocol was replaced with Na-dodecylsulfate). This DNA was used to transform the D-ribose-producing B. subtilis strain 31094 (U.S. Pat. No. 3,970,522) using the natural competence based method (Example 2). The transformants were screened by the PCR methods described in the Example 2 and also by studying the products of the glucose fermentation by these strains. One clone generating the fragment of the expected size in PCR with the oligonucleotide pair oBS-RPI5 and oBS-RPI3 and retaining the ability of the parent strains to convert glucose into five-carbon sugars was selected. The rpi-disrupted derivative of strain ATCC 31094 was named GX2.

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Abstract

The invention relates to the methods of manufacturing five-carbon sugars and sugar alcohols as well as other compounds derived from pentose-phosphate pathway from readily available substrates such a hexoses using metabolically engineered microbial hosts.

Description

[0001]This applications is a continuation-in-part of international application number PCT / FI01 / 00051, filed Jan. 22, 2001 pending, and a continuation-in-part of U.S. application Ser. No. 09 / 488,581, filed Jan. 21. 2000, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 08 / 790,585, filed Jan. 29, 1997, now U.S. Pat. No. 6,723,540, which is a continuation of U.S. application Ser. No. 08 / 368,395, filed Jan. 3, 1995, issued as (U.S. Pat. No. 5,631,150), which is a continuation of U.S. application Ser. No. 08 / 110,672, filed Aug. 24, 1993, abandoned, which is a continuation-in-part of U.S. application Ser. No. 07 / 973,325, abandoned, the contents of each of which are fully incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to the methods of manufacturing five-carbon aldo- and keto-sugars and sugar alcohols by fermentation in recombinant hosts. Especially, the invention is directed to recom...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12P19/02C12N1/15C12N1/19C12N1/21C07H1/00C07H3/02C12N9/04C12N9/10C12N9/12C12N9/90C12P7/18
CPCC07H1/00C07H3/02C12N9/0006C12N9/1022C12N9/1241C12N9/90C12P7/18
Inventor MIASNIKOV, ANDREIOJAMO, HEIKKIPOVELAINEN, MIRAGROS, HAKANTOIVARI, MERVIRICHARD, PETERRUOHONEN, LAURAKOIVURANTA, KARILONDESBOROUGH, JOHNARISTIDOU, ARISTOSPENTTILA, MERJAPLAZANET-MENUT, CLAIREDEUTSCHER, JOSEF
Owner DANISCO SWEETENERS
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